HLA alloantibodies and the mechanism of the antiglobulin-augmented lymphocytotoxicity procedure.
HLA Class I alloantigens express multiple epitopes which can be defined serologically using human HLA alloantibodies (aAb). We have shown that the vast majority of HLA antisera exhibit the CYNAP phenomenon (complement-dependent cytotoxicity (CDC) negative, adsorption positive) which can be identified by conversion to direct CDC positive reactivity with the addition of an antihuman immunoglobulin (Ig) light chain (AHG) reagent. In this study, the immunochemical mechanisms responsible for the CYNAP phenomena and how AHG overrides CYNAP have been further characterized using affinity-purified HLA aAb, class-specific anti-IgH reagents and human C1q binding assays quantified by flow cytometry. We have found that CYNAP reactions are not the result of low affinity aAb or generally caused by non-complement fixing HLA aAb. Our experiments illustrate that only anti-human IgL AHG reagents can consistently augment CDC and override CYNAP; anti-IgH have not effective. Two noncompeting HLA aAb of different epitopic specificity or one aAb in conjunction with the AHG-augmenting reagent results in striking synergy with a 200 to 400% increase in binding of C1q. We conclude from these and other experiments detailed in this article that an IgM aAb or either two adjacent, noncompeting IgG HLA aAb bound to spatially distinct epitopes on a single HLA molecule or a monospecific IgG HLA aAb in concert with the AHG binding to this HLA aAb, is required for efficient (bivalent) C1q binding and initiation of C-mediated lympholysis. In contrast, the CYNAP phenomenon usually occurs because monospecific HLA aAb directed against a single epitope cannot effect high affinity, bivalent interaction with Clq and activate complement that would ultimately lead to cytolysis.
Fuller
T C
TC
Department of Surgery, Massachusetts General Hospital, Boston, USA.
Fuller
A A
AA
Golden
M
M
Rodey
G E
GE
D000888:Antibodies, Anti-Idiotypic; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface; D001666:Binding Sites, Antibody; D003167:Complement Activation; D015922:Complement C1q; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D006680:HLA Antigens / Q000276:immunology*; D006650:Histocompatibility Testing; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007518:Isoantibodies / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D000954:Antigens, Surface; D006680:HLA Antigens; D007074:Immunoglobulin G; D007518:Isoantibodies; D015922:Complement C1q